A role for ethylene in the metabolism of cyanide by higher plants

The action of ethylene on the capacity of plant tissues to metabolize cyanide to β-cyanoalanine was examined. Beta-cyanoalanine synthase (EC 4.4.1.9) catalyzes the reaction between cyanide and cysteine to form β-cyanoalanine and hydrogen sulfide. Levels of β-cyanoalanine synthase activity in tissues of 6 day old etiolated pea (Pisum sativum) seedlings were enhanced severalfold by 1 microliter per liter ethylene. The promotive effect of ethylene increased with increasing ethylene concentrations from 0.01 to 100 microliters per liter and with the period of exposure from 3 to 24 hours. Ethylene enhanced β-cyanoalanine synthase activity in all regions of the seedling (shoots and roots, internodal regions, cotyledons). The promotive effect was eliminated by norbornadiene, a competitive inhibitor of ethylene action. Levels of β-cyanoalanine synthase in seedlings of four other dicots (Phaseolus aureas, Glycine max, Lactuca sativa, Sinapis arvensis) and two monocots (Hordeum vulgares, Triticum aestivum) were also increased in response to ethylene. Our results suggest an important regulatory role for ethylene in the metabolism of cyanide by higher plants.     

Effect of path or sink anoxia on sugar translocation in roots of maize seedlings

The subcellular and developmental distribution of β-cyanoalanine synthase (EC 4.4.1.9), which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, were investigated in barley (Hordeum vulgare) leaves. Total leaf activity was 1.1 micromoles per minute per gram fresh weight. Sucrose density gradients of lysed mesophyll protoplasts of barley revealed the exclusive or predominant localization of β-cyanoalanine synthase in the mitochondria. The enzyme was absent from both vacuole and chloroplast fractions.

β-Cyanoalanine synthase activity was distributed over the entire length of the barley leaf. Activity was dependent on the developmental stage, with a 3.5-fold higher activity in the oldest (apical) compared to the youngest (basal) parts of the leaf. The corresponding difference in activity for mesophyll protoplasts isolated from these parts was 7.5-fold. In younger leaf seagments, the nonchlorophyllous tissues accounted for up to 70% of the total β-cyanoalanine synthase activity. These results are discussed with reference to the formation of HCN as a substrate in barley leaves.

     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

Effect of 2,4-Dichlorophenoxyacetic Acid on Endogenous Cyanide, beta-Cyanoalanine Synthase Activity, and Ethylene Evolution in Seedlings of Soybean and Barley

Treatment of etiolated seedlings of barley (Hordeum vulgare) and soybean (Glycine max) with 1 millimolar 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in a 14-fold and greater than 100-fold increase in ethylene production, respectively. Simultaneous monitoring of endogenous cyanide and β-cyanoalanine synthase (β-CAS) (EC 4.4. 1.9) activity was also performed. Endogenous levels of cyanide did not change in barley. In soybean, endogenous cyanide increased within 3 hours, increased again 6 hours after exposure to 2,4-D, and continued to increase throughout the experimental period. The activity of β-CAS increased in both barley and soybean 9 hours after herbicide treatment. The increase in cyanide preceded the increase in β-CAS activity by 3 to 6 hours in soybean. The steady-state concentration of endogenous cyanide in soybean was 1 micromolar, based on rates of ethylene production and cyanide metabolism by β-CAS. This agreed with the determination of endogenous cyanide by both distillation and isotope dilution. Given the apparent compartmentalization of β-CAS in mitochondria and the localization of ethylene/HCN production at the plasmalemma and/or tonoplast, our results suggest that extra-mitochondrial accumulation of cyanide in the cytoplasm may occur. If so, the activity of cyanide-sensitive cytoplasmic enzymes could be adversely affected, thus possibly contributing to the toxicity of 2,4-D.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

Cyanide action in plants — from toxic to regulatory

Recent biochemical and genetic studies on hydrogen cyanide (HCN) metabolism and function in plants were reviewed. The potential sources of endogenous cyanide and the pathways of its detoxification are outlined and the possible signaling routes by which cyanide exerts its physiological effects are discussed. Cyanide is produced in plant tissues as the result of hydrolysis of cyanogenic compounds and is also released as a co-product of ethylene biosynthesis. Most cyanide produced in plants is detoxified primarily by the key enzyme β-cyanoalanine synthase. The remaining HCN at non-toxic concentration may play a role of signaling molecule involved in the control of some metabolic processes in plants. So, HCN may play a dual role in plants, depending on its concentration. It may be used in defense against herbivores at high toxic concentration and may have a regulatory function at lower concentration. Special attention is given to the action of HCN during biotic and abiotic stresses, nitrate assimilation and seed germination. Intracellular signaling responses to HCN involve enhancement of reactive oxygen species (ROS) generation and the expression of cyanide-insensitive alternative oxidase (AOX) and ACC synthase (ACS) genes. The biochemical and cellular mechanisms of these responses are, however, not completely understood.     

Dormancy removal in apple embryos by nitric oxide or cyanide involves modifications in ethylene biosynthetic pathway

The connection between classical phytohormone-ethylene and two signaling molecules, nitric oxide (NO) and hydrogen cyanide (HCN), was investigated in dormancy removal and germination “sensu stricto” of apple (Malus domestica Borkh.) embryos. Deep dormancy of apple embryos was removed by short-term (3–6 h) pre-treatment with NO or HCN. NO- or HCN-mediated stimulation of germination was associated with enhanced emission of ethylene by the embryos, coupled with transient increase in ROS concentration in embryos. Ethylene vapors stimulated germination of dormant apple embryos and eliminated morphological anomalies characteristic for young seedlings developed from dormant embryos. Inhibitors of ethylene receptors completely impeded beneficial effect of NO and HCN on embryo germination. NO- and HCN-induced ethylene emission by apple embryo was only slightly reduced by inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity during first 4 days of germination. Short-term pre-treatment of the embryos with NO and HCN modified activity of both key enzymes of ethylene biosynthetic pathway: ACC synthase and ACC oxidase. Activity of ACC synthase declined during first 4 days of germination, while activity of ACC oxidase increased markedly at that time. Additional experiments point to non-enzymatic conversion of ACC to ethylene in the presence of ROS (H₂O₂). The results indicate that NO and HCN may alleviate dormancy of apple embryos “via” transient accumulation of ROS, leading to enhanced ethylene emission which is required to terminate germination “sensu stricto”. Therefore, ethylene seems to be a trigger factor in control of apple embryo dormancy removal and germination.     

Dependence of in vivo ethylene production rate on 1-aminocyclopropane-1-carboxylic Acid content and oxygen concentrations

1-Aminocyclopropane-1-carboxylic acid (ACC) is aerobically oxidized in plant tissues to form ethylene by ethylene-forming enzyme (EFE). The effect of substrate (ACC and oxygen) concentrations on ethylene production rate by plant tissues was investigated. The K_m value for O₂ in ethylene production varied greatly depending on the internal ACC content. When ACC levels in the tissue were low (below its K_m value), the concentration of O₂ giving half-maximal ethylene production rate ([S]_0.5) ranged between 5 and 7%, and was similar among different tissues. As the concentration of ACC was increased (greater than its K_m value), [S]_0.5 for O₂ decreased markedly. In contrast, the K_m value for ACC was not much dependent on O₂ concentration, but varied greatly among different plant tissues, ranging from 8 micromolar in apple (Malus sylvestris Mill.) tissue to 120 micromolar in etiolated wheat (Triticum aestivum) leaf. Such a great variation was thought to be due to the different compartmentation of ACC within the cells in different tissues. These kinetic data are consistent with the view that EFE follows an ordered binding mechanism in which EFE binds first to O₂ and then to ACC.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.)

Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd²⁺ at concentrations above 1 micromolar. Cd²⁺-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd²⁺, which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca²⁺, present during a 2-hour preincubation, reduced the effect of Cd²⁺ on leakage and ACC conversion. This suggests that Cd²⁺ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.     

Chilling-Induced Ethylene Production in Cucumbers (Cucumis sativus L.)

1-Aminocyclopropane-1-carboxylic acid (ACC) level, ACC synthase activity, and ethylene production in cucumbers (Cucumis sativus L.) remain low while the fruit are held at a temperature which causes chilling injury (2.5°C) and increase rapidly only upon transfer to warmer temperatures. The increase in ACC synthase activity during the warming period is inhibited by cycloheximide but not cordycepin or α-amanitin. Our data indicate that the synthesis of ACC synthase, which results in increased ACC levels and accelerated ethylene production, occurs only upon warming, possibly from a message produced or unmasked during the chilling period. Ethylene production by chilled (2.5°C) cucumbers increased very little upon transfer to 25°C if the fruit were chilled for more than 4 days. The fruit held for 4 days or longer showed a large increase in ACC levels but little ethylene production even in the presence of exogenous ACC. This suggests that the system which converts ACC to ethylene is damaged by prolonged exposure to the chilling temperature. Cucumbers stored at a low but nonchilling temperature (13°C) showed very little change in ACC level, ethylene production, or ACC synthase activity even after transfer to 25°C.     

Hydrogen cyanide and embryonal dormancy in apple seeds

Embryos of apple ( Malus domestica Borh. cv. Antonówka) were treated with 1 m M gaseous HCN for 6 h and cultured under a 12 h photoperiod. HCN pretreatment stimulated germination, increased the length of hypocotyls, shortened the main root and decreased the percentages of seedlings with asymmetrically grown as well as with asymmetrically greened cotyledons. High activity of β-cyanoalanine synthase (EC 4.4.1.9) and a sharp increase in cyanogen content during embryo culture suggested very low levels of endogenous HCN. despite the activity of HCN releasing enzymes. The obtained data allow us to postulate an important role for cyanide in the regulatory complex controlling dormancy in apple seeds. Experiments with respiratory inhibitors indicated, however, that HCN pretreatment affected neither the alternative electron transport pathway nor residual respiration.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

Inhibition of ethylene production in fruit slices by a rhizobitoxine analog and free radical scavengers

The rhizobitoxine analog, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid (Ro), which effectively inhibits ethylene production in apple (Malus domestica Borkh.) and other tissues at concentrations at about 68 micromolar, inhibited ethylene production by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) fruit slices but only by about 15% in pink and ripe tomato tissue slices. Ethylene production in climacteric-rise and postclimacteric avocado slices was likewise relatively insensitive to 68 micromolar Ro. At 340 micromolar Ro, inhibition of ethylene production increased up to 50% in pink tomato slices, whereas 680 micromolar Ro was required to inhibit ethylene production by 30% in avocado slices. Incorporation of ¹⁴C from [¹⁴C]methionine into ethylene in green and pink tomato tissues was inhibited by Ro to about the same extent as inhibition of total ethylene production. Results thus far are inconclusive as to the mechanism of Ro resistance in tomato and avocado tissues. At 1 millimolar, free radical scavengers such as benzoate, propyl gallate, nordihydroguaiaretic acid, and to a lesser extent, eugenol, inhibited ethylene production in both Ro-sensitive (green tomato and apple) tissues and Ro-resistant (pink tomato and avocado) tissues. Therefore, free radical steps are suggested in the ethylene-forming systems.     

Mobilization and utilization of cyanogenic glycosides: the linustatin pathway

In the seeds of Hevea brasiliensis, the cyanogenic monoglucoside linamarin (2-β-d-glucopyranosyloxy-2-methylpropionitrile) is accumulated in the endosperm. After onset of germination, the cyanogenic diglucoside linustatin (2-[6-β-d-glucosyl-β-d-glucopyranosyloxy]-2- methylpropionitrile) is formed and exuded from the endosperm of Hevea seedlings. At the same time the content of cyanogenic monoglucosides decreases. The linustatin-splitting diglucosidase and the β-cyanoalanine synthase that assimilates HCN, exhibit their highest activities in the young seedling at this time. Based on these observations the following pathway for the in vivo mobilization and metabolism of cyanogenic glucosides is proposed: storage of monoglucosides (in the endosperm)—glucosylation—transport of the diglucoside (out of the endosperm into the seedling)—cleavage by diglucosidase—reassimilation of HCN to noncyanogenic compounds. The presence of this pathway demonstrates that cyanogenic glucosides, typical secondary plant products serve in the metabolism of developing plants as N-storage compounds and do not exclusively exhibit protective functions due to their repellent effect.     

Ethylene-Enhanced 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Ripening Apples

Apples (Malus sylvestris Mill, cv Golden Delicious) were treated before harvest with aminoethoxyvinylglycine (AVG). AVG is presumed to reversibly inhibit 1-aminocyclopropane-1-carboxylic acid (ACC) activity, but not the formation of ACC synthase. AVG treatment effectively blocked initiation of autocatalytic ethylene production and ripening of harvested apples. Exogenous ethylene induced extractable ACC synthase activity and ripening in AVG-treated apples. Removal of exogenous ethylene caused a rapid decline in ACC synthase activity and in CO₂ production. The results with ripened, AVG-treated apples indicate (a) a dose-response relationship between ethylene and enhancement of ACC synthase activity with a half-maximal response at approximately 0.8 μl/l ethylene; (b) reversal of ethylene-enhanced ACC synthase activity by CO₂; (c) enhancement of ACC synthase activity by the ethylene-activity analog propylene.

Induction of ACC synthase activity, autocatalytic ethylene production, and ripening of preclimacteric apples not treated with AVG were delayed by 6 and 10% CO₂, but not by 1.25% CO₂. However, each of these CO₂ concentrations reduced the rate of increase of ACC synthase activity.